Human Immunity to Malaria The first Kenieroba study (08-I-N120) was completed in 2012 and focused on: 1) Whether Malian children with various hemoglobin and red cell polymorphisms are protected from clinical Plasmodium falciparum malaria; 2) Developing a profile of the acquisition of malaria immunity in Malian children; 3) Examining aspects of pathogenesis due to malaria in Malian children and adults. To initiate these investigations, we conducted a 4-year longitudinal study of 1500 children ranging in age from 6 months to 18 years. All the enrolled children were typed for a series of these red cell and hemoglobin polymorphisms. After diagnosis and treatment of 4207 episodes of malaria, we have developed a very detailed profile of age-dependent acquisition of resistance to malaria in this population published in 2015. In addition, we continue to analyze a series of samples collected throughout the study, including using an antibody-dependent respiratory burst assay. We have also investigated standard hematological indices which are commonly used in malaria epidemiologic studies to calculate parasite densities. Red blood cell (RBC) count, hemoglobin (Hb) level, hematocrit (Ht), white blood cell (WBC) count, and WBC subsets were measured in paired blood samples from Malian children, and the data were stratified by month (May, January) and age group. Interestingly, the indices change during a malaria season and with increasing age, factors which need to be taken into account in epidemiologic studies of malaria (Diakite et al. AJTMH, 2016). An additional clinical study in Kenieroba was completed in 2015 in conjunction with Dr. Michael Walther, formerly of LMIV (13-I-N209).The primary objective was to demonstrate that co-administration of IV artesunate (AS) and oral activated charcoal (oAC) is not inferior to administration of IV AS alone, with regard to the parasite half-life in children presenting with uncomplicated malaria. To show this we enrolled 35 children into each of two arms, AS + oAC and AS + water. During two transmission seasons we enrolled 71 children with 70 completing the study. Initial comparative analysis of the two groups showed that there was no significant difference in parasite clearance rate and no difference in reportable adverse events. This data provides the basis for determining whether oAC co-administration with AS can reduce severe malaria in children. Malaria Transmission Transmission of malaria is a critical aspect of the parasite life cycle but is poorly understood, and we have increased our efforts to investigate parasite sexual stages, malaria transmission in the field, and the biology of the mosquito vector. In May of 2014 we completed a clinical protocol studying 500 individuals of all ages in Kenieroba (13-I-N107). Volunteers were finger-pricked twice per month for a year to analyze both DNA and RNA. This past year we analzyed parasite DNA by PCR directly on filter paper and we have completed testing of all the samples (>10,000). Using sensitive molecular methods rather than slide reading, we have found that a relatively high proportion of individuals of all ages carry malaria parasites prevalence was highest in November of 2013 during the wet season and lowest in May of 2014. Unlike most studies, we assessed longitudinal prevalence of parasites in the population and by a linear regression model, only age and gender showed significant effects on the longitudinal prevalence (p<0.0001 and p=0.0008, respectively), while other host factors did not. We showed for the first time that increasing P.falciparum longitudinal prevalence throughout the year was associated with decreasing risk of clinical malaria. This suggests that those with persistent parasite carriage acquire stronger protective immunity against clinical malaria. In addition, we have established methods for quantitation of gametocyte mRNA encoding Pfs25 using RT-PCR and we have determined that more than 80% of people with parasites also have detectable gametocytes throughout the year, showing that no one group can be uniquely targeted for interventions to reduce transmission. Naturally Occurring Wolbachia in Anopheline Mosquitoes from Mali Wolbachia is a bacteria commonly found in various arthropods and is being studied as a vehicle to control insect populations. We have established that Wolbachia infections occur in field-caught adult female Anopheles gambiae s.l. at two localities in Mali. Using real-time PCR techniques, the prevalence of Wolbachia infection in An. gambiae s.l. was 45% in Dangassa and 42.5% in Kenieroba. We have also shown that Wolbachia levels are significantly higher in field-collected female mosquitoes that are not infected with P. falciparum. Direct quantification of the levels of Wolbachia and the number of sporozoites present (based on genome copies present on the same sample) indicates that all mosquitoes with very high levels of sporozoites were not infected with Wolbachia, and that females with very high levels of Wolbachia did not carry sporozoites. These observations suggest that Wolbachia may be affecting malaria transmission. Wild female mosquitoes were collected in Dangassa and maintained in the insectary of MRTC/ICER Mali for individual oviposition. Both the mothers and the batch of eggs were sent to the Laboratory of LMVR/NIH/NIAID where colonies were established and the presence of Wolbachia was confirmed and has been maintained for 5 generations. A selection for progeny from Wolbachia-positive females is underway in Mali to establish a colony of Wolbachia positive mosquitoes. A Wolbachia-negative colony will be established from eggs of uninfected females that will also be treated with Tetracycline to completely eliminate Wolbachia. This will allow studies on the impact of Wolbachia on the mosquito immune system and its impact on P. falciparum infection. Dry Season Ecology of Malaria Vectors Our main work is focused on: (1) long distance migration of mosquitoes as means of re-colonization of Sahelian villages after population extinction, and (2) understanding the strategies of persistence of Anopheles coluzzii in the Sahel throughout the dry season. Building on previous results, we have amassed additional compelling evidence that A. coluzzii (previously, the M form of A. gambiae) persists locally by aestivation whereas A. gambiae (previously, the S form) and A. arabiensis re-colonize the area after the onset of rains. In our recent study on the desiccation tolerance of A. coluzzii we found a seasonal allometry manifesting as smaller thorax size compared with wing size and smaller spiracle length compared with both wing size and thorax size (Arcaz et al. 2016), probably reflecting an adaptation to minimize water loss, a distinct developmental modality leading to the production of aestivating adults from larvae. If this marker can reliably distinguish aestivating from non-aestivating mosquitoes, it will allow addressing many questions about aestivating mosquitoes. Our project on long distance migration combines (i) on-the-ground monitoring of vector density and composition along 9 surveillance villages, spanning over 400 km, with (ii) aerial sampling of mosquitoes using traps tethered to helium filled balloons. This year, we completed the aerial sampling and have moved to data analysis of this project. In addition we have established bio-assays to measure flight aptitude of mosquitoes aiming at identifying long-distance migrants on the ground. Monthly data testing over a year to follow seasonal variation in flight aptitude among mosquito species is nearly completed. After this years fieldwork, we plan to publish the first report of wind-assisted long distance migration of malaria vectors. These findings have important implications for malaria control and for the understanding and modeling of malaria.